RT Journal Article
SR Electronic
T1 Phosphorylation motif dictates GPCR C-terminal domain conformation and arrestin interaction
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.02.23.529712
DO 10.1101/2023.02.23.529712
A1 Myriam Guillien
A1 Assia Mouhand
A1 Amin Sagar
A1 Aurélie Fournet
A1 Frédéric Allemand
A1 Glaécia A. N. Pereira
A1 Aurélien Thureau
A1 Pau Bernadó
A1 Jean-Louis Banères
A1 Nathalie Sibille
YR 2023
UL http://biorxiv.org/content/early/2023/02/23/2023.02.23.529712.abstract
AB Arrestin dependent G protein-coupled receptor (GPCR) signaling pathway is regulated by the phosphorylation state of GPCR’s C-terminal domain, but the molecular bases of arrestin:receptor interaction are to be further illuminated. Here we investigated the impact of phosphorylation on the conformational features of the C-terminal region from three Rhodopsin-like GPCRs, the vasopressin V2 Receptor (V2R), the Growth Hormone Secretagogue or ghrelin Receptor type 1a (GHSR) and the β2-Adernergic Receptor (β2AR). Using phosphomimetic variants, we identified pre-formed secondary structure elements, or short linear motif (SLiMs), that undergo specific conformational transitions upon phosphorylation. Of importance, such conformational transition favors arrestin-2 binding. Hence, our results suggest a model in which the cellular signaling specificity of GPCRs is encoded in the phosphorylation-dependent structuration of the C-terminal regions, which will subsequently modulate arrestin conformation and therefore GPCR:arrestin signaling outcomes.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsIDPintrinsically disordered proteinIDRintrinsically disordered regionPTMpost-translational modificationsGPCRG protein-coupled receptorGRKG protein receptor kinaseV2Rvasopressin V2 receptorGHSRghrelin receptor 1aβ2ARβ2-adreneric receptorwt-V2R-Cterwild-type V2R C-terminuspm-V2R-Cterphosphomimetic V2R C-terminuswt-GHSR-Cterwild-type GHSR C-terminuspm-GHSR-Cterphosphomimetic GHSR C-terminuswt-β2AR-Cterwild-type β2AR C-terminuspm-β2AR-Cterphosphomimetic β2AR C-terminusV2Rppsynthetic fully phosphorylated peptide of V2R C-terminusC7ppsynthetic fully phosphorylated peptide of CXCR7 C-terminuspRho-Cterphosphorylated C-terminus of rhodopsin receptorPPIIpolyproline helix 2CDcircular dichroismMSmass spectrometryMSTmicroscale thermophoresisFRETfluorescence resonance energy transfertSAXSsmall angle X-ray scatteringSDS-PAGEsodium dodecyl sulphate-polyacrylamide gel electrophoresisSEC-MALSsize exclusion chromatography – multi angle light scatteringNMRnuclear magnetic resonanceSCSsecondary chemical shiftCSPchemical shift perturbationHSQCheteronuclear single quantum coherenceNOEnuclear Overhauser effectPREparamagnetic relaxation enhancementRDCresidual dipolar couplingFMflexible meccanoEDTAEthyleneDiamineTetraAcetic acidDTTdithiothreitolCouL-(7-hydroxycoumarin-4-yl ethylglycinePMSFphenylmethylsulfonyl fluorideTCEPTris(2-CarboxyEthyl)Phosphine